权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Regulation of heterotrimeric G protein signaling by subunit phosphorylation

通过亚基磷酸化调节异源三聚体 G 蛋白信号传导

基本信息

批准号：
10387072
负责人：
SARAH M ASSMANN
金额：
$ 7.75万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10387072
关键词：
Arabidopsis Award BODIPY Biochemical Biological Assay Biology Chemistry Clinical Communicable Diseases Data Development Disease Disease susceptibility Eukaryota Event Funding G-Protein Signaling Pathway GTP Binding GTP-Binding Protein alpha Subunits GTP-Binding Proteins Genetic Diseases Guanosine Triphosphate Health Heterotrimeric GTP-Binding Proteins Human Human Characteristics Hydrolysis In Vitro Intervention Laboratories Luciferases Measurement Mediating Methods Molecular Probes Mutation Parents Pathway interactions Pharmaceutical Preparations Phenotype Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Plant Model Plants Protein Subunits Proteins Reader Regulation Repression Research Research Personnel Signal Transduction Signaling Protein Speed System United States National Institutes of Health Validation Variant Work data acquisition design fluorophore instrumentation mutant protein protein interaction small molecule synergism

项目摘要

Summary Heterotrimeric G protein signaling pathways are of tremendous importance to human health. Mutation of G protein subunits causes genetic disease, developmental abnormalities, and altered infectious disease susceptibility. Indeed, G protein pathways are the targets of approximately a third of all drugs under clinical use. The Assmann laboratory has furthered fundamental understanding of heterotrimeric G protein signaling through elucidation of G protein-mediated signaling cascades and phenotypes. The emphasis of the parent award is on mechanisms of phospho-regulation of G protein signaling that are evolutionarily conserved but have been oft overlooked in mammalian systems. In this research, the model plant Arabidopsis is used as a facile system to investigate kinase-mediated phosphorylation of the canonical G protein α (Gα) subunit, GPA1, and to explicate the downstream signaling impacts of this phosphorylation; in particular, how phosphorylation biases interactions with downstream effector proteins. In parallel, relevance to human health is demonstrated through assessment of the impacts of analogous phosphorylation events on human Gα subunits in vitro, using BODIPY-GTP binding and hydrolysis assays on the corresponding human phosphomimic mutants. This supplement requests funding for the purchase of a BioTek Synergy Neo2 plate reader to increase the throughput and reliability of the BODIPY- GTP activity assays and to allow the implementation of orthogonal methods, particularly transcreener assays, that will allow independent validation of these biochemical data. The dual monochromators of the Synergy Neo2 plate reader will allow for assay versatility with a multitude of fluorophores. The speed and sensitivity of the Synergy Neo2 far outpaces that of the extant obsolete Flx800 plate reader, allowing for finer timescale measurements of a greater number of Gα variants. In addition, the capability of the Synergy Neo2 to assay luciferase activity will facilitate assessment of biased signaling arising from phosphorylation-dependent protein- protein interactions, as the Synergy Neo2 has the capability for high-throughput measurements of protein-protein interaction using the split-luciferase method. In summary, the proposed state-of-the-art plate reader will provide reliable, sensitive, and rapid data acquisition as well as entirely new capabilities to probe the molecular effects of G protein phosphorylation. Finally, the instrumentation will be available to other NIH-funded researchers at Penn State in the Biology and Chemistry Departments.

总结异源三聚体G蛋白信号通路对人类健康具有重要意义。G突变蛋白质亚基导致遗传疾病、发育异常和改变的感染性疾病易感性事实上，G蛋白途径是临床使用的所有药物中约三分之一的靶点。 Assmann实验室通过以下方法进一步加深了对异源三聚体G蛋白信号传导的基本理解：阐明G蛋白介导的信号级联和表型。家长奖的重点是 G蛋白信号的磷酸化调节机制在进化上是保守的，但经常被认为是一种新的机制。在哺乳动物系统中被忽视。本研究以拟南芥为模式植物，研究激酶介导的经典G蛋白α（Gα）亚基GPA 1的磷酸化，并解释这种磷酸化的下游信号影响;特别是，磷酸化如何偏向相互作用下游效应蛋白。与此同时，通过评估证明与人类健康的相关性类似的磷酸化事件在体外对人Gα亚基的影响，使用BODIPY-GTP结合和对相应的人磷酸模拟突变体的水解测定。该补充申请需要资金购买BioTek Synergy Neo 2酶标仪，以提高BODIPY的通量和可靠性- GTP活性测定并允许实施正交方法，特别是transscreener测定，可以独立验证这些生化数据Synergy Neo 2的双单色仪读板器将允许具有多种荧光团的测定通用性。的速度与灵敏度 Synergy Neo 2远远超过现存的过时Flx 800酶标仪，允许更精细的时间尺度更多Gα变体的测量。此外，Synergy Neo 2测定荧光素酶活性将有助于评估由磷酸化依赖性蛋白引起的偏置信号传导，蛋白质相互作用，因为Synergy Neo 2具有高通量测量蛋白质-蛋白质相互作用的能力。使用分裂荧光素酶方法进行相互作用。总之，所提议的最先进的读板器将提供可靠、灵敏和快速的数据采集以及全新的探测分子效应的能力 G蛋白的磷酸化。最后，仪器将提供给其他NIH资助的研究人员，宾夕法尼亚州立大学生物和化学系。